EP1697511A2 - Compositions d'immunotherapie, methodes de preparation et d'utilisation de ces dernieres - Google Patents

Compositions d'immunotherapie, methodes de preparation et d'utilisation de ces dernieres

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Publication number
EP1697511A2
EP1697511A2 EP04821259A EP04821259A EP1697511A2 EP 1697511 A2 EP1697511 A2 EP 1697511A2 EP 04821259 A EP04821259 A EP 04821259A EP 04821259 A EP04821259 A EP 04821259A EP 1697511 A2 EP1697511 A2 EP 1697511A2
Authority
EP
European Patent Office
Prior art keywords
composition
fadd
poly
dsrna
microparticle
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP04821259A
Other languages
German (de)
English (en)
Other versions
EP1697511A4 (fr
Inventor
Russell G. Higbee
Glen N. Barber
Anatoly M. Kachurin
Olga M. Kachurina
Heather Gappa-Fahlekamp
William L. Warren
Siddharth Balachandran
Emmanuel Thomas
Robert Parkhill
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
VaxDesign Corp
Original Assignee
Sciperio Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sciperio Inc filed Critical Sciperio Inc
Publication of EP1697511A2 publication Critical patent/EP1697511A2/fr
Publication of EP1697511A4 publication Critical patent/EP1697511A4/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/117—Nucleic acids having immunomodulatory properties, e.g. containing CpG-motifs
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    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
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    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
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    • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
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    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
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    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1641—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poloxamers
    • A61K9/1647—Polyesters, e.g. poly(lactide-co-glycolide)
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    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1652—Polysaccharides, e.g. alginate, cellulose derivatives; Cyclodextrin
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    • A61K9/167—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction with an outer layer or coating comprising drug; with chemically bound drugs or non-active substances on their surface
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    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1135—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against oncogenes or tumor suppressor genes
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Definitions

  • the present invention relates to the field of immunotherapy. More particularly, it relates to compositions capable of activating either or both the
  • compositions are particularly useful in modulating innate immune responses
  • a host exposes to microbial pathogens such as viruses, bacteria, and fungi that triggers the activation of innate immune responses that galvanize early host defense mechanisms as well as invigorate adaptive immune responses involving cytotoxic T cell activity and antibody production [Medzhitov, et al, Semin. Immunol, 10:351-353, (1998)].
  • pathogenic microbes and the triggering of the innate immune cascade has become the subject of intense research over the past few years.
  • TLRs Toll-like receptors
  • PAMPs pathogenic microorganisms
  • Figure 1 pathogen-associated molecular patterns - PAMPs
  • the TLRs were first identified in Drosophila (the fruit fly) and have been demonstrated as playing an important role in fly development as well as in host defense against fungi and gram-positive bacteria [Imler, et al., Curr. Top. Microhiol Immunol, 270:53-79, (2002)] .
  • TLR Engagement of a TLR transmits a signal to the cell's nucleus, inducing the cell to begin producing certain proteins such as cytokines, alerting other components of host defenses.
  • cytokines proteins
  • LPS extracellular lipopolysaccharide
  • dsRNA dsRNA .[Takeda, et al., Ann. Rev. Immunol, 21 :335- 376, 2003].
  • signaling pathways are initiated through homophilic interactions triggered by a Toll/interleukin (IL)-l receptor (TIR) domain present in the cytosolic region of all TLRs [Akira, Jour.
  • IL Toll/interleukin
  • TLRs including TLR-2, -4, and -5, use a common adaptor protein referred to as MYD88, which contains a TIR domain as well as a death domain (DD).
  • MYD88 common adaptor protein
  • DD death domain
  • TRJJF/TICAM, TRAM, and TIRAP/Mal have now been isolated and similarly function in the modulation of TLR activity [Horng, et al., Nat. Immunol, 2:835-841, (2001); Oshiumi, et al, Nat.
  • IRAK IL-1 receptor-associated kinase
  • IRAK-1 and -4 which are DD-containing serine-threonine kinases involved in the phosphorylation and activation of TRAF-6 [Cao, et al., Science, 271:1128-1131, (1996); Ishida, et al., J. Biol Chem., 271:28745-28748, (1996); Muzio, et al, Science, 278:1612-1615, (1997); Suzuki, et al., Nature, 416:750-756,
  • TLRs trigger common signaling pathways that culminate in the activation of the transcription factors NF- ⁇ B as well as the mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK), p38, and c- Jun N-terminal kinase (JNK) [Akira, J. Biol. Chem. , 278 :38105-38108, (2003)] .
  • MAPKs mitogen-activated protein kinases
  • ERK extracellular signal-regulated kinase
  • JNK c- Jun N-terminal kinase
  • TLR-3 or -4 can activate the transcription factor interferon regulatory factor (IRF)-3, perhaps through TRIF-mediated activation of the noncanonical I ⁇ B kinase homologues, I ⁇ B kinase-e (IKKe), and TANK-binding kinase- 1 (TBK1), although the exact mechanisms remain to be clarified [Doyle, et al, Immunity, 17:251 -263, (2002); Fitzgerald, et al., Nat. Immunol. , 4:491 -496,
  • Activation of the NF- ⁇ B, ERK/JNK, and IRF-3 responsive signaling cascades culminates in the transcriptional stimulation of numerous genes that regulate the innate and adaptive immune responses including the inflammatory response.
  • Activation of primary innate immune response genes such as IFN- ⁇ induces not only anti-viral genes, but also molecules that facilitate innate immune responses involving NK cells, the maturation of DCs as well as upregulation of chemokines and molecules such as MHC that facilitate T-cell responses.
  • IFN has also been shown to be critically important for the production of antibody responses.
  • Particle carriers have been devised to deliver drugs, antigens and other signal molecules to cells [Aideh, et al., J. Microencapsul, 14:567-576 (1997); Akbuga, et al., Microencapsul, 13:161-167 (1996); Akbuga, et al., Int. J. (1994); Aral, et al., STP Pharm. Sci., 10:83-88 (2000)]. Requirements of these delivery carriers differ depending on application.
  • chemokines need to provide stable gradients of the loaded molecules for an extended period of time (usually days) and the particles need to be relatively large (200-700 ⁇ m) to avoid being phagocytosed.
  • immunization is stronger when antigens are carried by smaller particles that not only interact with cells via their surface, but can also be engulfed by dendritic cells, macrophages or other antigen presenting cells (APCs).
  • Phagocytosis is optimal for the particles smaller than 10 ⁇ m, which stipulates sizes for antigen carriers.
  • Chitosan is a natural product derived from chitin. It is chemically similar to cellulose, which is the major composition of plant fiber, and possesses many properties as fiber. Chitosan has been shown to exhibit high adhesion to mucosa
  • chitosan has shown promise as a carrier for delivery
  • the composition comprises: a microparticle
  • the modulator of FADD-dependent pathway is selected from the group consisting of double-stranded RNA (dsRNA), poly(IC), a component of the FADD-dependent pathway, a DNA plasmid encoding a component of the FADD-dependent pathway, a bacterium, and a fungus.
  • the modulator of TLR pathway is selected from the group consisting of dsRNA, poly (IC), a synthetic mimetic of viral dsRNA, and a ligand for TLR, a bacterium, and a fungus.
  • the microparticle is further coated with a targeting molecule that binds specifically to an antigen presenting cell.
  • a composition for modulating immune system in a host comprising phagocytosable chitosan microparticles loaded with a nucleic acid and a protein.
  • a method for treating viral, bacterial, fungal infection and cancer in a subject comprising administering to said subject an effective amount of the composition described above.
  • Yet another aspect of the present invention relates to a method for preparing a multifunctional microparticle for immune modulation.
  • the method comprises the steps of fabricating chitosan microparticles by precipitation, gelation and spray; and incubating the chitosan microparticles in a solution comprising a nucleic acid, a protein, or both.
  • Another aspect of the invention relates to creating particles with « multiple/multifunctional agents that can activate both innate and adaptive immune responses.
  • Yet another aspect of the present invention relates to a method for
  • FADD-deficient cells comparing to poly (IC)-treated wild-type cells.
  • FIGURES Figure 1 illustrates the detection of PAMPs by a host cell through TLRs.
  • Figure 2 illustrates the antiviral mechanism of interferons.
  • Figure 3 is a schematic of the TNF- ⁇ pathway.
  • Figure 4 is a schematic of the pathways of antigen processing and delivery
  • FIG. 5 is a schematic of Poly(IC) treatment protocol.
  • Figure 6 is a schematic of the proposed method of enhancing innate
  • Figure 7 is a structural formulation of chitosan.
  • Figure 8 is a microscopic picture showing polystyrene beads phagocytosed by a monocyte-derived human dendritic cell.
  • Figure 9 is a structural formula of branched PEL
  • Figure 10 is the artificial virus-like particles consisting of (1) yeast dsRNA, (2) spermidine-polyglucin-glutathione conjugate, and (3) hybrid protein TBI-GST.
  • Figures 1 la-1 If are experimental data showing that FADD, but not caspase-8, is required for prevention of NSN replication in MEFs even after IF ⁇ pretreatment.
  • Figure 11a shows that FADD-deficient MEFs are susceptible to NSN- induced CPE despite IF ⁇ pretreatment and photomicrographs were taken 48 hours post-infection.
  • Figure 1 lb shows that FADD-deficient MEFs are not protected from NSN-triggered cell death by IF ⁇ pretreatment.
  • Figure l ie shows that IF ⁇ pretreatment delays, but does not prevent, NSN replication in FADD -/- EFs.
  • Figure lid shows that caspase-8 deficiency does not predispose MEFs to increased susceptibility to NSV induced CPE.
  • Figure l ie shows that caspase-8 +/+ and -/- MEFs are equally well-protected from VSV- induced cell death by IF ⁇ pretreatment.
  • Figure 1 If shows that IF ⁇ pretreatment efficiently inhibits VSV replication in both caspase-8 +/+ and -/- EFs.
  • Figures 12a-12d are experimental data illustrating that absence of FADD sensitizes cells to the infection by encephalomyocarditis virus (EMCV) and influenza virus (FLU) infection.
  • Figure 12a shows that FADD is required to protect against EMCV-induced CPE. Cells were photographed (Mag. 200x) 24 hours post infection.
  • Figure 12b shows that cells infected as in (a) were analyzed for cell viability by Trypan Blue exclusion.
  • Figure 12c shows that FADD is required to protect against EMCV-induced CPE. Cells were photographed (Mag.
  • Figure 12d shows that cells infected as in (c) were analyzed for cell viability by Trypan Blue exclusion.
  • Figures 13a-13f are experimental data illustrating that IF ⁇ signaling is not disrupted in FADD -/- MEFs.
  • Figure 13a shows normal STAT1 phosphorylation in the absence of FADD.
  • Figure 13b shows that nuclear translocation of STAT1 following IFN treatment occurs normally in the absence of FADD.
  • Figure 13c shows that FADD is not required for IFN-triggered gene induction.
  • Figure 13d shows IFN-responsive promoters function normally in the absence of FADD.
  • Figure 13e shows that exogenous IFN- ⁇ can protect FADD -/- MEFs from VSV- induced CPE when added after infection.
  • Figure 13f shows exogenous IFN- ⁇ can protect FADD -/- MEFs from VSV replication and consequent cell death when added after infection.
  • Figures 14a and 14b are experimental data illustrating that De Novo synthesis of IFN- ⁇ is required to afford continued protection of wild type MEFs following VSV infection despite IFN- ⁇ / ⁇ pretreatment.
  • Figure 14a shows that FADD +/- cells are susceptible to VSV in the presence of neutralizing anti-IFN- ⁇ antiserum despite IFN- ⁇ / ⁇ pretreatment. Photographs were taken 48 hours post infection (mag. 200x).
  • Figure 14b shows that FADD +/- cells treated as in (a) were examined for VSV progeny yield or cell viability by Trypan Blue exclusion.
  • Figures 15a-15g are experimental data illustrating that defective IFN- ⁇ gene induction by intracellular dsRNA in the absence of FADD.
  • Figure 15a shows that transfected dsRNA-mediated activation of the IFN- ⁇ promoter is defective in FADD -/- MEFs.
  • Figure 15b shows that dsRNA-induced production of IFN- ⁇ is defective in the absence of FADD.
  • Figure 15c shows that reconstitution of murine (M) FADD into FADD -/- MEFs can partially rescue dsRNA signaling
  • Figure 15d shows that caspase-8 is not required for intracellular dsRNA signaling.
  • Figure 15e shows that PKR is not required for intracellular dsRNA signaling.
  • PKR +/+ and PKR -/- MEFs were transfected with IFN- ⁇ -Luc.
  • Figure 15f shows that RNAi-mediated knockdown of FADD, but not PKR or TLR3 abolishes intracellular dsRNA signaling.
  • Figure 15g shows that
  • FIGS. 16a-16e are experimental data showing that TRL3 signaling does
  • FIG. 16a shows that TLR3 and other TLR signaling components induce IFN- ⁇ normally in FADD -/- MEFs.
  • Figure 16b shows that
  • TRAF6 deficiency does not predispose MEFs to VSV infection in the presence of
  • Figures 17a-17f are experimental data showing that RIP deficiency mimics FADD ablation.
  • Figure 17a shows that RJP-deficient EFs are very susceptible to
  • Figure 17b shows that RlP-deficient EFs are not protected from VSV-triggered cell death by IFN pretreatment.
  • Figures 17d and 17e show the defective intracellular dsRNA
  • Figures 18a-18j are experimental data illustrating that the antiviral pathway incorporating FADD signals via TBK-1/IKK- ⁇ and IRF-3.
  • Figure 18a shows infection of wild-type or IKK- ⁇ -, IKK- ⁇ -, IKK- ⁇ - and IKK- ⁇ -deficient MEFs with VSV (MOI y 4 10) with or without IFN- ⁇ / ⁇ (100 Uml21) pre-treatment.
  • Figure 18b is the DNA microarray analysis of a selected set of antiviral genes.
  • Figure 18c shows IFN- ⁇ production after transfection with poly(I:C), or treatment with poly(I:C) alone.
  • Figure 18d shows IFN- ⁇ production after transfection with the indicated amounts of poly(I:C), or treatment with ⁇ oly(I:C) alone.
  • Figure 18e is the localization of IRF-3 after transfection of po!y(I:C) for 1 h in FADD +/- and FADD -/- cells.
  • Figure 18f is the defective IRF-3- responsive promoter activation in FADD -/- MEFs.
  • Figure 18g is the infection of Irf3 +/+ and Irf3 -/- MEFs with VSV (MOI % 10) with or without IFN- ⁇ / ⁇ (100 Uml21) or IFN- ⁇ (0.5 ng ml21) pre-treatment.
  • Figure 18h shows IFN- ⁇ production after transfection with poly(I:C), or treatment with poly(I:C) alone.
  • Figure 18i shows IFN- ⁇ production after transfection with poly(I:C), or treatment with poly(LC) alone.
  • Figure 18j is the DNA microarray analysis for a selected set of antiviral genes. Error bars indicate mean ⁇ s.d.
  • Figures 19a- 19c are experimental data illustrating that FADD -/- Cells are susceptible to infection by gram-positive and gram-negative intracellular bacteria.
  • Figure 19a shows that FADD -/- cells are very susceptible to CPE induced by intracellular Listeria infection.
  • Figure 19b shows that FADD -/- cells are susceptible to cell death induced by intracellular Listeria infection.
  • Figure 19c shows that FADD -/- cells are very susceptible to CPE induced by intracellular
  • Figure 20 is a Modified Electrospray device with turbulent receiver.
  • Figure 21 is an ESEM image of Chitosan Microparticles prepared by Modified Electrospray with turbulent agitation.
  • Figure 22 is a structural formula for polyinosinic-polycytidylic acid, poly(IC).
  • Figure 23 is a structural formula for Ethidium Homodimer.
  • Figure 24 shows a calibration curve for measuring poly(IC) by fluorescence of intercalated Ethidium Homodimer.
  • Figures 25a and 25b show a comparison of measuring loose and bound poly(IC) using intercalating Ethidium Homodimer intercalator.
  • FIG. 26 shows time dependent fluorescent of the chitosan particles loaded with poly(IC) upon their interaction with Ethidium Homodimer.
  • Figure 27 shows time release of poly(IC) from the chitosan microparticles.
  • Figures 28a and 28b show purple complexes of monovalent copper with proteins and Bicinchoninic Acid.
  • A is Biuret complex with peptide nitrogens.
  • B is chelate complex with Bicinchoninic Acid.
  • Figure 29 shows a calibration curve for the Bicinchoninic Acid assay of Ovalbumin.
  • Figure 30 represents time release of Ovalbumin from the chitosan microparticles.
  • Figures 31a and 31b are the SEM images of freeze dried Protasan poly(IC) particles. A is supra-micron size particles, XI 00. The bar shows 200 ⁇ m.
  • B is sub micron size particles, X5000.
  • the bar shows 5 ⁇ m.
  • Figures 32a-32c are the sorption of poly(IC) by supra-micron protasan particles at different pH.
  • A shows the optical spectra of poly(IC) decreasing as a result of sorption.
  • FIG. 33a and 33b are the sorption properties of PLGA/PEI particles.
  • A shows the sorption of poly(IC) for the particles obtained by different
  • Figures 34a and 34b illustrate PLGA/PEI/poly(IC) particles obtained via
  • A is SEM X5000, after solubilization; and B shows sorption capacity: affected by solubilization at high or low ionic
  • Figures 35a and 35b show the particles of PLGA/PEI/poly(IC).
  • A is the SEM image X5000;
  • FIG. B is the fluorescent micrograph of diluted water suspension, X200.
  • Figure 36 illustrates the induction of IFN ⁇ and IFN ⁇ in DC1 and DC2 subsets of human dendritic cells by PLGA/PEI particles with poly(IC).
  • Figures 37a and 37b show the extracellular TLR 3 induction via microparticles with poly (IC).
  • Figure 38 illustrates that DC2 subsets in peripheral human blood samples were exposed to PLGA/PEI or Protosan particles (with or without amalgamated dsRNA) and monitored for IFN ⁇ expression after 3-6 hours of exposure to the particles
  • the present invention provides methods and compositions for modulating innate immune responses to antigens.
  • the composition contains an activator for the fas-associated death domain molecule (FADD)/RIP dependent pathway.
  • FADD fas-associated death domain molecule
  • the signaling pathway incorporating FADD was found to be Toll-Like-Receptor (TLR)-independent and therefore, FADD plays an essential role in innate immunity to viral infection by functioning in the recognition of intracellular dsRNA species, which is critical for the induction of key antiviral responses, including the production of Type I IFN, and that FADD is also involved in the recognition of other pathogens such as bacteria and fungi.
  • TLR Toll-Like-Receptor
  • Traditional antigen- presenting cells include, but not limited to macrophages, dendritic cells, langerhans cells, and B lymphocytes. Follicular dendritic cells are also considered to be
  • the "innate immune response” is the way the body recognizes and defends
  • the innate immune response functions as a first
  • NK cells natural killer cells
  • mast cells mast cells
  • eosinophils eosinophils
  • soluble molecules collectively known as acute phase proteins, such as the interferons, specific components of the complement cascade and cytokines, that serve to
  • lymphocytes In comparison to innate immunity, adaptive immunity develops when the
  • a “cellular immune response” is one mediated by T lymphocytes and/or other white blood cells.
  • T lymphocytes and/or other white blood cells One important aspect of cellular immunity involves an antigen-specific response by cytotoxic T lymphocytes
  • CTLs have specificity for peptide antigens that are presented in
  • MHC major histocompatibility complex
  • CTLs help induce and promote the destruction of intracellular microbes, or the lysis of cells infected with such
  • antigen refers to any agent (e.g., any substance,
  • immunogen refers to any agent (e.g., any substance, compound, molecule [including macromolecules], or other moiety)
  • protein molecules or at least one portion of a protein molecule, which contains one or more epitopes encompasses protein molecules or at least one portion of a protein molecule, which contains one or more epitopes.
  • antigens are also immunogenes,
  • antigen is often used interchangeably with the term “immunogen.”
  • immunogen is often used interchangeably with the term “immunogen.”
  • the substance may then be used as an antigen in an assay to detect the presence of
  • tumor-specific antigen(s) refers to antigens that are present only in a
  • a melanoma-specific antigen is an antigen that is expressed only in melanoma cells but not in normal melanocytes.
  • a major consequence of viral infection includes the activation of primary innate immune response genes such as IFN- ⁇ .
  • the production of IFN- ⁇ induces not only anti-viral genes, but also molecules that facilitate immune responses involving NK cells, the maturation of DCs as well as upregulation of chemokines and molecules such as MHC that facilitate T-cell responses.
  • intracellular and extracellular dsRNA utilize divergent signaling pathways to induce IFN- ⁇ .
  • intracellular dsRNA species generated as a consequence of virus replication are recognized through a TLR-independent, FADD-related pathway.
  • the viral dsRNAs are recognized by an intracellular receptor molecule, which recruits FADD and RIPl into an 'innateosome' complex to activate the NF- ⁇ B, ERK/JNK, and IRF-3 pathway.
  • Activation of the NF- ⁇ B, ERK/JNK, and IRF-3 responsive signaling cascades leads to the expression of numerous genes that regulate the innate and adaptive immune responses including the inflammatory response.
  • the extracellular PAMPs including dsRNA and LPS, are recognized through a TLR-related pathway that also leads to the activation of the NF- ⁇ B, ERK/JNK, and IRF-3 responsive signaling cascades.
  • TLR-related pathway that also leads to the activation of the NF- ⁇ B, ERK/JNK, and IRF-3 responsive signaling cascades.
  • both the FADD-dependent and TLR-dependent pathways are also involved in the recognition of other pathogens such as bacteria and fungi (see e.g., Imler et al.
  • MHC class I/peptide complexes are ligands for T-cell receptors (TCRs) of CD8 T cells.
  • Extracellular foreign antigens are taken into intracellular vesicles, endosomes. As the pH in the endosomes gradually decreases, proteases are activated that digest antigens into peptide fragments. After fusing with vesicles that contain MHC class II molecules, antigenic peptides are placed into the antigen-binding groove. Loaded MHC class II/peptide complexes are transported to the cell surface, where they are recognized by the TCRs of CD4 T cells. Further, as shown in Figure 4, extracellular or exogenous antigens are phagocytozed by DCs which then localize these antigens to the lysosomal compartment where proteolytic enzymes digest and process the antigen.
  • the antigen is then moved to the cellular surface on class II MHC molecules and never is in the cytosol of the DC.
  • soluble proteins present in the cytosol of the DC are continuously degraded by proteasomes.
  • These antigenic molecules are combined with class I MHC in the endoplasmic reticulum which move them to the cell surface via vesicles.
  • the strict dichotomy between MHC I and MHC II pathways was challenged by several studies that have shown that peptides generated from exogenous proteins can gain access to the cytosol and therefore be presented on class I MHC molecules [Roake, et al., J. Exp.
  • antigen delivered in a particulate form either absorbed to solid polymer microspheres [Raychaudhuiri, et al., Nat. Biotechnol. 16:1025-1031, 1998], encapsulated in microspheres [Maloy, et al., IMMUNOLOGY, 81:661-667, 1994], or aggregated in the form of immunocomplexes with antibody [Rodriguez, et al,. Nat. Cell Biol, 1:362-368, 1999], triggers an efficient "cross-presentation" pathway that allows the antigen to be loaded on class I MHC.
  • compositions for modulating innate immune responses that are capable of cross- signaling both the intracellular and extracellular pathways.
  • the compositions may trigger the "cross-presentation" pathway that allows the antigen to be loaded on class I MHC and allows the development of an immune reaction against viral or malignant tumor antigens before the viral infection or tumor formation takes place .
  • the composition contains a first modulator for the intracellular FADD-dependent signaling pathway and a second modulator for extracellular TLR-independent signaling pathway.
  • the modulators are loaded onto a chitosan-based microparticle that can be phagocytozed by a professional APC such as a DC.
  • the term "loaded” refers to the association of the activators to the microparticle, either by encapsulation or by surface attachment.
  • modulators of FADD-dependent signaling pathway include, but are not limited to, dsR ⁇ A, poly (IC), synthetic mimetic of viral dsR ⁇ A, components of FADD-dependent pathway such as FADD and RIPl, D ⁇ A encoding a component of FADD pathway, as well as bacteria, fungi, and other antigens that are known to activate or suppress FADD-dependent pathway.
  • modulators of TLR-dependent signaling pathway include, but are not limited to, TLR ligands such as dsRNA, poly (IC), synthetic mimetic of viral dsRNA, and LPS; components of TLR-dependent pathway such as MYD88,
  • a modulator of the FADD-dependent pathway may also function as a modulator of the TLR-dependent pathway. Therefore, the first modulator and the second modulator in the composition of the present invention can be the same molecule.
  • a dsRNA molecule may activate both the FADD-dependent pathway and the TLR-dependent pathway. If the dsRNA encodes a suppressor for FADD-dependent pathway, the same molecule may activate the TLR-dependent pathway while suppressing the FADD-dependent pathway.
  • the present invention also provides methods for identifying antiviral, antibacterial, and anti-fungal genes induced through FADD signaling pathway using FADD-/- and FADD+/+ cells.
  • Figure 5 depicts one embodiment for identifying antiviral gene induced through FADD signaling pathway. Briefly, FADD-/- and FADD+/+ cells are treated with poly (IC). RNA isolated from the treated cells is hybridized to a DNA array of genes to determine dsRNA-induced genes. The expression levels of the dsRNA-induced genes are further confirmed by
  • RNA interference is developed to inhibit the expression of dsRNA-induced genes and the susceptibility to viral infection in the RNAi-treated cells is examined.
  • RNAi is a phenomenon of the introduction of dsRNA into certain organisms and cell types causes degradation of the homologous mRNA. RNAi was first discovered in the nematode Caenorh ⁇ bditis elegans, and it has since been found to operate in a wide range of organisms. In recent years,
  • RNAi has becomes an endogenous, efficient, and potent gene-specific silencing technique that uses double-stranded RNAs (dsRNA) to mark a particular transcript for degradation in vivo.
  • dsRNA double-stranded RNAs
  • RNA f technology is disclosed, for example, in U.S. Patent No. 5,919,619 and PCT Publication Nos. WO 99/14346 and WO 01/29058.
  • the first and second modulators of the composition of the present invention are the same dsRNA.
  • the dsRNA loaded microparticles would bind TLR and activate the TLR-dependent signaling pathway.
  • the dsRNA-loaded microparticles would be phagocytozed (by macrophages, DCs, monocytes) and activate FADD-dependent signaling pathway.
  • the dsRNA encodes an immune activator.
  • the dsRNA is opened and translated to produce the immune activator that further activates the innate immune pathway.
  • the dsRNA may encode a component of the TLR pathway, such as TRIF or the IRAKs, which when introduced into cells would augment TLR-mediated activation of IFN- ⁇ and other innate immune responses.
  • the first modulator is dsRNA and the second
  • modulator is a component of the TLR pathway or a DNA molecule encoding a
  • the first modulator is a component of FADD-dependent pathway, such as FADD, or a DNA molecule encoding a component of
  • the second modulator is a dsRNA.
  • the first and second modulators are dsRNAs or
  • dsRNA messenger RNA
  • the dsRNA containing microparticles can be further coated with a ligand for TLR3 to activate the TLR3 pathway or with heat
  • shock proteins like gp96 or VSV G protein in order to target professional APCs
  • the microparticles can be loaded with dsRNA representing silencing RNAi (siRNA) that can target genes for suppression following engulfment.
  • siRNA silencing RNAi
  • the siRNA suppresses the expression of a component of the FADD-dependent pathway, such as FADD, and down
  • composition contains self-replicating RNA
  • RNA constructs bicistronic consisting of 5' terminal ORFs important for replicon IRES function and contains a
  • vims or other pathogens can be placed downstream of a second IRES.
  • the Replicon can be loaded onto chitosan particles and used to target antigen specific
  • the replicon will translate the foreign gene to produce antigen that can be processed tlirough the MHC class I or II pathways to stimulate CD4 and CD8 cells, specific
  • Replicons may be used to co-express pro-apoptotic molecules, such as caspases, or be co-loaded with purified pro-apoptotic molecules to induce
  • the chitosan particles loaded with intracellular or
  • extracellular FADD or TOLL activating molecules such as dsRNA (as described above) can be co-loaded with purified antigens, such as from influenza virus or other pathogen related molecules, which may become processed to stimulate CD4, CD8 cells.
  • the present invention utilizes polycationic microparticles as the delivery system for the modulators of FADD-dependent and TLR-dependent pathway.
  • Chitines and chitosanes are biodegradable polymers bearing multiple amino groups which acquire positive charges at neutral pH via association of hydrogen ion ( Figure7). Comparing to microparticles made of other polymers, chitosan-based microparticles provide decreased agglomeration and better loading capacity for negatively charged molecules, especially nucleic acids. Protasan, a more purified version of chitosan, will be used interchangeably herein.
  • the microparticles of the composition of the present invention are designed to achieve a three-fold objective: delivery, temporary protection from the
  • the microparticle of the present invention are designed to release or expose the associated RNA DNA/protein molecules quickly after entering the target cell to provide a vigorous immune response. In some applications, however, it may be desirable to release the associated molecules, such as cytokines, in a time-dependent manner.
  • cytokines examples include, but are not limited to, IL-12, IL-l ⁇ , IL-l ⁇ , D -15, IL-18, IFN ⁇ , IFN ⁇ , IFN ⁇ , IL-4, IL-10, IL-6, IL-17, IL-16, TNF ⁇ , and MIF; as well as chemokines such as MIP-3 ⁇ , MlP-l ⁇ , MIP-1 ⁇ , RANTES, MIP-3 ⁇ ,
  • Chitosan microparticles can be produced using methods known in the art.
  • Ravi Kumar et al. [Ravi Kumar, et al, Biomaterials, in press, 2003] demonstrated chitosan-stabilized PLGA cationic nanoparticles carrying DNA on their surfaces; the DNA was bound by simple mixing from watery solutions, thus, preserving integrity and conformation of the molecules.
  • standard emulsion technique involving vigorous mixing with the carrier solution and emulgation schemes is also suitable for chitosan encapsulation of plasmids along with protein antigens [Thiele, et al., J.
  • Controlled Release 76:59-71, 2001].
  • These protocols can be utilized to prepare particles carrying various sets of cytokines or heat shock proteins together with dsRNA and/or DNA plasmids as discussed earlier.
  • Preferred methods for producing small microparticles are the micro gun and modified electrospray techniques, which are described in more details in the Examples.
  • the "crumpled paper" shape enabled these particles with high surface areas for a high adsorption capacity for proteins and nucleic acids.
  • Chitosan polymers can be cross-linked with a crosslinking agent.
  • crosslinking agents include, but are not limited to inorganic polyions, such as tripolyphosphate (TPP), sodium sulphate, and organic agents, such as glutaraldehyde and genipin.
  • TPP tripolyphosphate
  • organic agents such as glutaraldehyde and genipin.
  • Loading of nucleic acid and/or protein in chitosan particles can be achieved by direct admixing the nucleic acid and/or protein with chitosan during the fabrication of microparticles, externally saturating prefabricated microparticles with the nucleic acid and/or protein solutions, or a combination thereof.
  • the external saturation method provides a higher loading efficiency than the direct admixing method. Combination of the two methods, however, showed an synergistic effect in enhancing the loading efficiency.
  • the microparticles of the present invention is small enough to be effectively phagocytosed and processed by APCs such as DCs and macrophages, as well as their precursors such as monocytes.
  • the size of the microparticle is in a range from 0.5 to 70 microns, and more preferably from 0.5 to 20 microns.
  • Figure 8 shows polystyrene beads, 4.5 ⁇ m, phagocytosed by monocyte-derived human DCs [(Thiele et al., J cont. release 76:59-71 (2001)].
  • the phagocytic properties of the microparticles is modified by using a mixture of hydrophilic chitosan polymer and one or more hydrophobic polymers.
  • Nano-sizes of chitosan particles may be produced using methods described in the examples.
  • chitosan particles up to hundreds of micrometers, can be synthesized using the protocol of Denkbas et al. [Denkbas, et al., Reactive & Functional Polymers, 50:225-232, (2002)].
  • the release rates of nucleic acid and/or protein from chitosan particles can be controlled by adjusting several factors including the molecular weight of chitosan, the degree of deacetylation of chitosan, and the weight/charge ratios between chitosan and loaded biomolecules.
  • the chitosan-based dsRNA/DNA/protein loaded micropaticles is encapsulated within a poly(lactide-co-glycolide) (PLGA) matrix/microparticles containing cytokines or antigens.
  • PLGA poly(lactide-co-glycolide)
  • cytokines or antigens cytokines or antigens.
  • PLGA has been shown to be biocompatible and it degrades to toxicologically acceptable lactic and glycolic acids that are eventually eliminated from the body. Release rates of the cytokines and the chitosan particles could be further controlled by adjusting the parameters involved for PLGA encapsulation, including monomer ratio/molecular weight of PLGA.
  • chitosan/Protasan is hydrophilic
  • the uptake of the particles into the cell across the cell membrane may be enhanced.
  • other types of polymers may be incorporated into the chitosan-based microparticles to achieve variable release profiles for the loaded biomolecules.
  • a hydrophobic polymer such as PLGA
  • PLGA can be blended with the more hydrophilic chitosan to form cationic PLGA particles.
  • suitable polymers include, but are not limited to, poly(caprolactone), poly (oxybutirate) .
  • poly(ethylene imine) PEI
  • PEI poly(ethylene imine)
  • Figure 9 branched amphiphilic polyamine
  • Another example includes forming porous particles by the addition of polyanionic sodium alginate to polycationic chitosan, as described by Liu et al. [Liu, et al.,k J. Controlled Release, 43:65-74, 1997]. By adjusting the ratio of the polymers, the pore size could be controlled and therefore the release rates of the dsRNA/cytokines from the particles.
  • the present invention also contemplates using cationic liposomes as a delivery vehicle.
  • Cationic liposomes are good carriers for RNA, DNA and peptides [Honda, et al., J Virol. Metk, 58:41-58, 1996; Nastruzzi, et al, J.
  • liposomes offer a more adequate protection and better stabilization for RNA along with reasonable release kinetics.
  • the considerations regarding phagocytosis, surface charge, and hydrophilicity remain applicable to liposomes.
  • dsRNA and its immunogenic substitutes such as poly(IC) or poly(ICLC) can be encapsulated in the vesicles and/or be attached to the surface.
  • Phagocytosis of lipid cationic particles can be more pronounced than for hydrophilic colloid chitosan particles thanks to hydrophobic nature of the liposome surface. Special attention will be paid to controlling the appropriate 1-5 ⁇ m size of the lipid particle to enhanced phagocytosis.
  • liposome carriers are used for stimulating the internal FADD pathway via phagocytosis, whereas large chitosan microparticles is used as surface carriers exposing dsRNA to the surface TLRs. Many combinations can be envisaged.
  • Figure 10 shows an artificial vims-like particles comprises (1) yeast dsRNA, (2) spermidine- polyglucin-glutathione conjugate, and (3) hybrid protein TBI-GST.
  • a reverse particle is created with the dsRNA on the surface and a protein antigen in the center.
  • cross-signaling innate immune pathways is achieved with bacteria or fungus encapsulated in microparticles that undergo phagocytosis.
  • TLR pathway influences host defense against gram-positive bacteria while the imd (FADD) pathway exerts activity against gram-negative bacteria and fungus.
  • FADD imd
  • cross-signaling innate immune pathways is achieved with a tumor antigen or a polynucleotide encoding a tumor antigen encapsulated in microparticles that under go phagocytosis.
  • the preferred embodiments of the compounds and methods of the present invention are intended to be illustrative and not limiting. Modifications and variations can be made by persons skilled in the art in light of the above teachings. It is also conceivable to one skilled in the art that the present invention can be used for other purposes of measuring the acetone level in a gas sample, e.g. for monitoring air quality.
  • composition of the present invention is
  • infectious diseases include, but are not limited to, diseases caused by vimses, such as Human immunodeficiency vims (HIV); influenza vims (INV);
  • EMCV encephalomyocarditis vims
  • VSV stomatitis vims
  • parainfluenza vims EMCV
  • EMCV encephalomyocarditis vims
  • VSV stomatitis vims
  • parainfluenza vims parainfluenza vims
  • rhinovirus hepatitis A vims; hepatitis B vims; hepatitis C vims; apthoviras; coxsackievims; Rubella vims; rotavirus; Denque vims; yellow fever vims;
  • vims vims; varicello virus; Cytomegalovirus; variolavims; Vacciniaviras; suipoxvims and coronavims.
  • infectious diseases include, but are not limited to,
  • Campylobacter consisus Campylobacter recta; Candida albicans;
  • Pasteurella multocida Plasmodium falciparum; Porphyromonas gingivalis; Prevotella intermedia; Pseudomonas aeruginosa; Rothia dentocarius; Salmonella typhi; Salmonella typhimurium; Serratia marcescens; Shigella dysenteriae; Streptococcus mutants; Streptococcus pneumoniae; Streptococcus pyogenes; Treponema denticola; Trypanosoma cruzi; Vibrio cholera; and Yersinia
  • the composition of the present invention is administered into a mammal for the treatment of a cancer.
  • cancer include, but are not limited to, breast cancer, colon-rectal cancer, lung cancer, prostate cancer, skin cancer, osteocarcinoma, and liver cancer.
  • the present invention further relates to a pharmaceutical composition comprising a FADD activator and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may alternatively be administered subcutaneously, parenterally, intravenously, intradermally, intramuscularly, transdermally, intraperitoneally, or by inhalation or mist-spray delivery to lungs.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), or suitable mixtures thereof, and/or vegetable oils, solid microparticle or liposomes.
  • Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • the solution should be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, intratumoral and intraperitoneal administration.
  • sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage may be dissolved in 1 ml of isotonic NaCI solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (for example, "Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580).
  • isotonic NaCI solution for example, 1 ml of isotonic NaCI solution
  • hypodermoclysis fluid for example, 1 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (for example, "Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580).
  • Some variation in dosage will necessarily occur depending on the condition of the subject being treated.
  • the person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
  • preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the microparticles of the present invention may also be administered into the epidermis using the Powderject System (Chiron,
  • compositions disclosed herein may be formulated in a neutral or salt
  • Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic
  • acids such as, for example, hydrochloric or phosphoric acids, or such organic acids
  • Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases
  • bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like.
  • phrases "pharmaceutically-acceptable” or “pharmacologically- acceptable” refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human.
  • the preparation of an aqueous composition that contains a protein as an active ingredient is well understood in the art.
  • such compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared.
  • therapeutically effective amount as used herein, is that amount achieves, at least partially, a desired therapeutic or prophylactic effect in an organ or tissue.
  • the amount of the FADD activator necessary to bring about prevention and/or therapeutic treatment of the FADD deficiency related diseases (such as infectious diseases and cancers) or conditions is not fixed per se.
  • An effective amount is necessarily dependent upon the identity and form composition employed, the extent of the protection needed, or the severity of the diseases or conditions to be treated.
  • the present invention is further illustrated by the following examples which should not be constmed as limiting. The contents of all references, patents and published patent applications cited throughout this application, as well as the Figures and Tables are incorporated herein by reference.
  • EXAMPLE 1 FADD deficient fibroblasts are susceptible to vims infection It is observed that murine embryonic fibroblasts (MEFs) that lacked FADD appeared super sensitive to vims infection [Balachandran, et al., J. Virol, 74:1513- 1523, 2000]. To further examine this phenotype , a detailed analysis of vims replication in FADD +/- and FADD -/- MEFs using the IFN sensitive, prototypic rhabdovims vesicular stomatitis virus (VSV) was performed.
  • VSV prototypic rhabdovims vesicular stomatitis virus
  • FADD exerts a role in host defense against vims infection, a further investigation
  • FADD +/- and -/- MEFs were treated with either IFN ⁇ / ⁇ (500 U/ml) or IFN- ⁇ (5 mg/ml) for the indicated times, and STATl phosphorylation status determined by immunoblotting using a STATl phospho-tryosine 701 -specific antibody.
  • FADD +/- and -/- MEFs were transfected with a plasmid encoding a GFP-STAT1 fusion protein. 24 hours post-transfection, cells were treated with or without INF ⁇ / ⁇ (500 U/ml) or IFN- ⁇ (5 mg/ml) for one hour and STATl localization was determined by GFP fluorescence microscopy.
  • FADD +/- and -/- MEFs were treated with or without IFN ⁇ / ⁇ (500 U/ml) or IFN- ⁇ (5 mg/ml) for 18 hours. Lysates prepared from these cells were subject to immunoblot analysis for the indicated IFN-induced proteins. Similarly, the expression of selected type I and II IFN-induced genes including IRF-1, PKR and STAT2 in response to IFN, appeared unaffected in FADD -/- cells [Der, et al., Proc. Natl. Acad. Sci. USA, 95:15623-15628, 1998].
  • luciferase reporter genes under control of type I IFN (ISRE) or type II (GAS) exhibited normal activity when transfected into FADD -/- cells treated with IFN ( Figure lOd).
  • FADD +/- and FADD -/- MEFs were transfected with plasmids expressing luciferase under the control of either the interferon stimulated response element (ISRE-Luc) or the interferon gamma activate sequence (GAS-Luc). 24 hours later, cells were stimulated with or without IFN ⁇ / ⁇ (500 U/ml) or IFN- ⁇ (5 ng/ml) and luciferase activity measured 18 hours post treatment.
  • vims infection might explain the susceptibility of FADD -/- cells to vims
  • FADD +/- and FADD -/- cells were transfected with a luciferase reporter constmct under control of an IFN- ⁇ promoter
  • +/- and FADD -/- MEFs were transfected with a plasmid encoding luciferase under control of the human IFN- ⁇ promoter (IFN- ⁇ -Luc). 24 hours later, these cells were treated with poly(IC) alone [50 ⁇ g/ml], transfected poly(IC) [4 mg/ml in Lipofectamine2000) or LPS (5 ml/ml) and luciferase activity measured 6 or 24 hours post treatment. Data indicated that transfected ⁇ oly(IC) triggered robust (>10 fold) induction of the IFN- ⁇ promoter in FADD +/- cells but not in cells
  • FADD +/- and FADD -/- MEFs were treated with poly(IC) alone [50 ⁇ g/ml], transfected poly(IC) [4 mg/ml mg/ml in Lipofectamine2000) or LPS (5
  • RNAi-mediated knockdown of FADD, but not PKR or TLR3 abolishes intracellular dsRNA signaling.
  • HeLa cells were treated with siRNA sequences from mFADD, hFADD, PKR, or TLR3, and knockdown of the respective gene products confirmed by immunoblotting and RT-PCR (data not shown). These cells were then transfected with IFN- ⁇ -Luc, and subsequently transfected with poly(IC) (4 mg/ml in Lipofectamine 2000). Luciferase activity was measured 6 hours later.
  • PKR-deficient mice infected with VSV retained the robust ability to induce IFN- ⁇ (Figure 15). Neither could the observed virus/dsRNA-mediated activity be explained through TLR3 signaling. For example, we found little TLR3 activity in MEFs, HeLa and 293T cells ( Figures 15f-g). iRNA -mediated depletion of only FADD, and not PKR or TLR3 (or both simultaneously), in HeLa cells resulted in an almost complete abrogation of IFN- ⁇ promoter activity, in response to transfected poly(IC) ( Figure 15g). In Figure 15g, HeLa or TLR3 were transfected with a plasmid encoding TLR3, and expression was confirmed by flow cytometry (left).
  • VSV or poly(IC) to individually activate each of apical signaling cascades involved in IFN- ⁇ promoter activation, i.e. NF- ⁇ B, AP-1 and IRF-3 was examined [Agalioti, et al., Cell, 103:667-678, 2000; Thanos, et al.,
  • FADD-mediated signaling involves activation of NF- ⁇ B and
  • TLR3 is involved in the recognition of extracellular dsRNA, which can lead to the induction of IFN- ⁇ through activation
  • FADD +/- or FADD -/- MEFs were transfected with an IFN- ⁇ - luciferase reporter construct and plasmids encoding various components of the TLR signaling pathway (such as TLR3, IRAK-M, IRAK-1, MyD88, TIRAP/MAL, TRIF/TICAM-1, and TRAF6), many of which have been shown to induce IFN- ⁇ gene expression following transient overexpression [Akira, J. Biol. Chem., 278:38105-38108, 2003]. However, no abrogation in TLR-mediated induction of IFN- ⁇ was observed in FADD deficient cells ( Figure 16a).
  • IFN intracellular poly(IC)
  • Cell viability was determined by Trypan Blue exclusion analysis 48 hours post infection. This analysis indicated that transfected poly(IC) retained the ability to activate IFN- ⁇ in the absence of TRAF6, indicating that this adaptor molecule probably does not play a role in FADD-mediated dsRNA-intracellular signaling ( Figures 16c-d). Furthermore, it was not observed a significant role for FADD in other TLR pathways (data not shown).
  • FIG. 16e shows that IFN Pretreatment protects TRAF6 -/- MEFs from VSV.
  • the medium was examined for progeny virion presence 48 hours post-infection by standard plaque assay on BHK cells.
  • TRAF6 +/+ and TRAF6 - /- EFs were transfected with IFN- ⁇ -Luc for 24 hours, and subsequently transfected with poly(IC) (4 mg/ml in Lipofectamine 2000) for 6 hours, after which luciferase activity was measured.
  • TLR3 and IRAK-1 require TRAF6 for IFN- ⁇ gene induction.
  • TRAF6 +/+ and TRAF6 -/- EFs were transfected with plasmids encoding TLR3, IRAK-1 or TRAF6, along with IFN- ⁇ -Luc, and luciferase activity was measured 24 hours post-transfection.
  • EXAMPLE 5 A mammalian IMD-like pathwav confers anti-viral innate immunity Data indicate that FADD plays a key role in innate immunity to vims infection and is independent of the TRAF6 mediated TLR3 pathway. Further, FADD has recently reported to be involved in the innate immune response to bacterial infection in Drosophila [Leulier, et al., Curr. Biol, 12:996-1000, 2002;
  • the immunodeficient (imd) gene product a Drosophila homologue of the mammalian death domain containing kinase, RIP, associates with dFADD to trigger activation of anNF- ⁇ b related pathway and subsequent induction of antibacterial genes [Hoffmann, N twre, 426:33-38, 2003].
  • Figure 17a shows VSV-induced cytolysis in RIP -/- cells but not controls.
  • the medium was examined for progeny virion production.
  • RIP-deficient MEFs, as well as HeLa cells in which RIP expression was abrogated using RNAi exhibited a selective and profound inability to respond to intracellular dsRNA-mediated signaling of the IFN- ⁇ promoter
  • IRAK-1 or TRAF6, along with IFN- ⁇ -Luc, and luciferase activity was measured 24 hours post-transfection.
  • TLR3, IRAKI, TRAF6 and TRIF were able to robustly induce IFN- ⁇ promoter activity, following transient overexpression in RIP -/- MEFs, providing further evidence that intracellular and extracellular dsRNAs utilize divergent signaling pathways to induce IFN- ⁇ .
  • imd and dFADD are required to stimulate the induction of antimicrobial gene expression through activation of the NF- ⁇ B homologue Relish via an I- ⁇ B kinase (IKK) complex comprised of IKK- ⁇ /IRD5 and IKK- ⁇ /Kenny.
  • IKK I- ⁇ B kinase
  • induction of IFN- ⁇ also involves activation of NF- ⁇ B, as well as IRF-3.
  • IKK- ⁇ -, IKK- ⁇ -, IKK- ⁇ - and IKK- ⁇ - deficient MEFs were infected with VSV (MOI l A 10) with or without IFN- ⁇ / ⁇ (100 Uml21) pre-treatment.
  • Result shows that pre-treatment with IFN was able to effectively protect MEFs lacking IKK- ⁇ , - ⁇ or - ⁇ against vims infection (Figurel 8a).
  • RT-PCR and ELISA analyses confirmed a severe impairment of dsRNA- responsive induction of type I IFN, as well other antiviral genes, in the absence of
  • IRF-3 translocation which occurs after phosphorylation by TBK-1 /IKK- ⁇ and IKK-1,
  • viral dsRNAs are recognized by an intracellular receptor molecule, which may recruit FADD and RIPl into an 'innateosome'
  • TBK-1/IKK- ⁇ -deficient MEFs display a more profound defect in the induction of type I IFNs in response to dsRNA stimulation than either FADD-deficient or RIPl- deficient MEFs alone, plausibly suggesting that intracellular dsRNA-activated complexes retain some activity in the absence of FADD, or that alternative FADD- independent intracellular signaling cascades converge on TBK-1 /IKK- ⁇ .
  • This RIP 1 /F ADD/TBK- 1 (RIFT) pathway seems to be largely independent of TLR3 ,
  • EXAMPLE 6 The role of FADD in mammalian responses to bacterial infection The role of the imd pathway in Drosophila is reported to involve the response to gram -positive bacteria infection and the existence of an antiviral pathway has not yet been determined. Whether innate responses to intracellular bacterial infection that was effected by loss of FADD or RIP in mammalian cells was examined and as shown in Figure 19.
  • FADD +/-, FADD -/- or RIP -/- MEFs were treated with or without IFN- ⁇ / ⁇ or IFN ⁇ for 18 hours and infected with 5 ⁇ l of an over night culture of the intracellular gram-positive bacteria, Lysteria monocytogenes and incubated for a further 24 hours in medium containing 10 ig/ml gentamycin ( Figures 19a and 19b); or infected with 50 ⁇ l of an over night culture of the gram-negative Salmonella typhimurium, and incubated for a further 48 hours in medium containing 10 ig/ml gentamycin ( Figure 19c).
  • EXAMPLE 7 Prefabrication of chitosan particles with large surface area
  • Either a Micro Spray Air Gun or Electrospray methods were used for chitosan microparticles prefabrication.
  • the chitosan solution was dispersed turbulently to the smallest dimensions possible for the gun.
  • the sizes of the particles were controlled mostly by the surface tension and were in the range from ⁇ 20 to 100 microns.
  • Electrospray is a method of electrostatic atomization of liquids. An electrostatic field compels a fluid to jet out of a capillary electrode towards the receiving counter electrode. Secondary stepwise splitting and pulverization of droplets due to Coulomb repulsion produces plume of fine microdroplets.
  • a Modified Electrospray method was set up. Electrospraying of chitosan onto a still surface of the crosslinking solution (tripolyphosphate, TPP) resulted in the formation of thin surface film of the stabilized chitosan instead of microparticles, due to extremely fine and homogeneous pulverization of the chitosan solution. To prevent this undesirable effect, a turbulent recirculation of the crosslinking solution was devised ( Figure 20). A circulation micropump provided open loop circulation of the TPP solution in the receiving electrode plate essential for dismption of the film.
  • TPP tripolyphosphate
  • Electrospray unit was used to pulverize 1%, 1.5% and 2% chitosan solutions in water and 25% ethanol.
  • a 25G stainless steel capillaries (EFD) worked as pulverizing electrodes, while a 10 inch stainless steel plate containing 100 ml of 10% TPP solution was used as the receiving counter electrode.
  • Electrospray with the turbulent agitation of the crosslinking TPP solution created microparticles smaller than that obtained using the Micro Spray Gun plume mode: the sizes have occurred distributed from ⁇ 5 to ⁇ 50 microns ( Figure 21).
  • Chitosan droplets prefabricated by the modified electrospray method were of an around micron size: significant 90 degree scattering of red laser beam by the Electrospray plume was observed indicating to the droplet sizes comparable with the wavelength of light.
  • the larger apparent size observed for the dry particles is explained by their subsequent transformation: upon contact with the TPP solution the surface tension forces spread the microdroplets into the ultrathin sheets on the surface of TPP. This unusual shape was well seen in the microscope. Upon freeze drying the microsheets shrank into shapes resembling crumpled paper, and never spread again after re-suspending.
  • the above described methods of prefabrication microparticles produce wide range of the microparticles with large surface areas. The particles of the smaller size could be engulfed by dendritic cells. On the other hand, the large surface area of these particles provides a significant advantage for external saturation with nucleic acids and proteins.
  • EXAMPLE 8 Chitosan particles loaded with polyinosinic-polycytidylic acid
  • Polyinosinic-polycytidylic acid, poly(IC) is an interferon (IFN) inducer consisting of a synthetic, mismatched double-stranded RNA.
  • the polymer is made of one strand each of polyinosinic acid and polycytidylic acid ( Figure 22). Being a polyanion, poly(IC) is strongly adsorbed by the polycationic chitosan.
  • Time dependent fluorescence of the poly(IC) particles in the presence of ETDH has demonstrated two distinct phases: immediate intercalation of the easily accessible surface poly(IC) molecules accompanied by fast (a few seconds) buildup of fluorescence, and steady increase of the fluorescence due to slow penetration of ETDH deep in the particles. It has been considered necessary to obtain particles with maximal surface loading, i.e. demonstrating enhanced fast buildup of fluorescence. The following tentative order of efficiency has been found for the protocols of preparation of the chitosan/poly(IC) particles:
  • the easily accessible surface molecules of poly(IC) in the best particles prepared using Electrospray has comprised 4.7 ⁇ g poly(IC) per 1 mg of particles, which was - 12 times higher than for the particles prepared using Micro Gun by direct admixing (graphs 7 and 2 in Figure 26, respectively).
  • Particles prepared by direct admixing of poly(IC) to chitosan solution, 10 mg dry weight were placed in 1 ml of PBS in a plastic test tube, sealed and incubated on shaker at 37°C for 9 days.
  • OVA/poly(IC) loaded chitosan particles were used: Admixing to the bulk chitosan
  • Bicinchoninic Acid assay of OVA Bicinchoninic Acid (BCA) assay of proteins is based on two main steps: • The first step is a Biuret reaction which reduced Cu +2 to Cu +1 ; • In the second step Bicinchoninic Acid (BCA) substitutes peptide groups in the Biuret complex to form a bis-chelate complex with Cu +1 which is purple colored and detectable at 562 nm ( Figure 28).
  • BCA kits e.g. Sigma-Aldrich, Cat. # BCA1 usually contain BCA, Tartrate/Bicarbonate buffer (pH 11.25), and 4% copper sulfate solution.
  • OVA in solution has been measured as follows: Aliquots of protein solutions were added to the necessary excesses of the assay solution to guarantee the final optical extinction no more than 2, and a linear response of the assay altogether. The analytes were incubated for 1 hour in a rocker at 37°C. All readings were corrected to the reading of the bank sample containing zero protein. OVA in microparticles has been measured as follows: Samples of dry microparticles (about 1 mg each) were weighed on the analytical scaled (Mettler-Toledo XS105) with 0.01 mg accuracy and suspended in a corresponding excess of the BCA assay solution precalculated as to provide linear response and acceptable optical density ( ⁇ 2 o.u.).
  • test tubes were incubated either in the rocker for 4 hours at 37°C, or in water bath for 1 hour at 60°C with occasional tumbling. In both cases, the incubation times were determined experimentally to provide complete reducing of divalent copper to monovalent copper by molecules of the protein.
  • the tubes were centrifuged at 500 g for 5 minutes, to separate particles, and clear colored solutions were read on a
  • Modified Electrospray and Micro Spray Gun allow for prefabrication of
  • the high surface areas provide the large external surfaces for the NA and
  • TPP tripolyphosphate
  • Supra micro i.e. big
  • submicron small Protasan particles loaded with poly(IC) were prepared.
  • chemokine or dmg carriers used as chemokine or dmg carriers, or to activate extracellular TLR-3 immunity pathway; they should avoid being engulfed by cells.
  • immunization is known to be effective when nucleic acids and antigens are carried by smaller particles (0.5 to 10 um) that can be
  • EXAMPLE 12 Supra-micron Protasan particles highly loaded with polyinosinic-
  • the particles were washed in distilled water 6 times using recursive centrifugation / resuspension procedure and freeze dried overnight. The resulting
  • Amersham preparation was about 49 - 50%, all other components being buffering salts.
  • the amount of poly(IC) in the particles was calculated as the difference between the poly(IC) added to the system, and poly(IC) remaining in the aqueous phase after particle precipitation [Bivas-Benitz, et al, Int. J. Pharm., 266:17-27 (2003)].
  • concentration of poly(IC) in solution direct reading of poly(IC) UV spectra has been used instead of fluorescence methods, due to very high concentration of poly(IC) involved in the preparations. 2.
  • Measuring sorption of poly(IC) by prefabricated particles Particles of a known weight were suspended in a volume from 0.5 to 5 ml of acetate or phosphate buffer containing from 50 to 700 ug/ml poly(IC) in different experiments. The suspensions were vortexed intensively for 5 minutes, and then kept vortexed at intermediate level or rocked for another 15 minutes. Afterwards, the suspensions were centrifuged at 7000 g for 5 minutes, and the concentration of poly(IC) in the supernatant was measured in spectrophotometer using a 1-cm quartz cell.
  • the concentration of poly (IC) was determined using the difference of the optical absorptions at 260 and 400 nm, where every 1 optical unit corresponded to - 50 ⁇ g/ml poly(IC) (American Biosciences, specification for the Product #27-4732). 3. Sorption capacity of the supra micron Protasan particles as it depends on pH Solution of poly(IC) 0.7 mg/ml was mixed 1 : 1 with three different buffers:
  • Protasan Poly(IC)/Crosslinker agglomerates from diluted solutions.
  • 200 ml of poly(IC) solution, 200 ⁇ g/ml in 0.1% acetate buffer pH 4.5 was being added by drops within 15 minutes to 200 ml of Protasan solution 200 ⁇ g/ml in 0.1% acetate buffer at constant stirring at room temperature.
  • the resulting solution was stirred for 1 hour at 30°C, afterwards 400 ml of 10% solution of sodium sulfate was added by drops within 15 minutes.
  • the final 800 ml of the combined solution was stirred for 2 hours at 30°C, and then precipitated by centrifuging at 5000G.
  • the combined solution was poured dropwise in 50 ml 10% NaCI water solution kept under constant sonication in Branson-1510 sonication bath at room temperature.
  • Sodium chloride has been introduced to facilitate dispersing the organic phase and resuspending during the washing procedure.
  • the mixture was being sonicated for another 4 hours at elevated temperature (50°C) to eliminate the volatile solvents.
  • the resulting PLGA/PEI particles were washed /sedimented 4 times, as described before, and freeze dried. It was found possible to reduce the number of washing passes due to elimination of persistent surfactants.
  • Air Gun and Electrospray of PLGA/PEI solutions over NaCI receiving water solution The above described 5:1 PLGA: PEI solution in CH 2 Cl 2 /acetone was sprayed over 10% NaCI using Micro Air Gun and Electrospray over turbulent 10% NaCI solution. The collected microparticles were washed / sedimented 4 times and freeze dried. Sorption of poly(IC) by the particles obtained without surfactants has been tested as described above in the poly(IC) solution - 70 ⁇ g/ml.
  • EXAMPLE 15 Cross-signaling defense pathways against non- viral pathogens
  • the cross-signaling strategies may be useful in combating bacterial as well as vims-related disease.
  • OT-1 transgenic mouse that expresses the T cell receptor (TCR) for chicken ovalbumin (OVA) will be used as a model.
  • TCR T cell receptor
  • OVA ovalbumin
  • OVA containing particles are inoculated into animals (i.p.). This method was recently shown to demonstrate increased cross-presentation of OVA from gp96 expressing cells, to OVA-specific T-cells. By using this approach,
  • microencapsulation strategies that involve stimulation of the innate immune
  • Example 16 Demonstration that PLGA/PEI or Protasan microparticles loaded with poly (IC) induces INF ⁇ production in 293 cells bv activating the extracellular TLR3
  • Example 17 Demonstration that PLGA/PEI or Protasan microparticles loaded with poly (IC) induces INF ⁇ production in DC2 subset cells bv most likelv activating the intracellular innateosome pathwav DC2 subsets in peripheral human blood samples were exposed to PLGA/PEI or Protosan particles (with or without amalgamated dsRNA) and monitored for Interferon alpha expression after 3-6 hours of exposure to the particles as shown in Figure 38.
  • DC2 plasmacytoid DCs lack TLR 3 and so IFN alpha induction is being triggered by alternate dsRNA signaling pathways), most likely utilizing the intracellular pathway via the "innateosome.”
  • the preferred embodiments of the compounds and methods of the present invention are intended to be illustrative and not limiting. Modifications and variations can be made by persons skilled in the art in light of the above teachings. It is also conceivable to one skilled in the art that the present invention can be used for other purposes of measuring the acetone level in a gas sample, e.g. for monitoring air quality. Therefore, it should be understood that changes may be made in the particular embodiments disclosed which are within the scope of what is described as defined by the appended claims.

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Abstract

La présente invention concerne des compositions et des méthodes de modulation du système immunitaire. Un aspect de la présente invention se rapporte à une composition comprenant des modulateurs de la voie de signalisation dépendant du domaine effecteur de mort associé à Fas (FADD). Un autre aspect de la présente invention concerne des microparticules biodégradables, telles qu'une particule de chitosane ou une microparticule PLGA/PEI, conçue pour apporter des acides nucléiques et/ou des protéines, tels que des modulateurs de la voie de signalisation dépendant de FADD, pour amplifier/relancer différentes voies d'une réponse immunitaire. Un autre aspect de la présente invention se rapporte à une méthode de préparation de microparticules biodégradables. Le dernier aspect de cette invention porte sur l'utilisation du chitosane et d'autres microparticules polycationiques pour apporter des modulateurs de la voie de signalisation dépendant de FADD afin de moduler le système immunitaire en vue de prévenir et/ou de traiter des maladies infectieuses et des cancers.
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